Detection of cells producing murine interferon‐α using antipeptide antibodies

Abstract

Summary The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-a. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-a-1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH-Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-a molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-a, This finding is in agreement with the recent demonstration of constitutive IFN-a production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.

Role of interferons in the regulation of cell proliferation, differentiation, and development

“…IFN mRNA for certain subtypes was detected in spleen kidney, liver, and PBMC of normal individuals using S1 mapping (Tovey et al, 1987) and, more recently, reverse transcriptase-PCR (RT-PCR) (Brandt et al, 1993). Immunohistochemical studies will polyclonal, monoclonal or antipeptide antibodies has been used to identify IFN-a production in PBMC, histocytes of spleen, liver, and brain of normal individuals, normal mouse spleens, and uninduced lymphoid cell lines (Jilbert et al, 1986;Greenway et al, 1992;Khan et al, 1989;Hwang et al, 1994). These direct demonstrations of IFN production support earlier evidence based on the regulation of the levels of IFN-induced enzymes and effects of anti-IFN antibodies in vivo and on cultured cells that there is a constitutive production of type I IFNs that probably act in a local paracrine or autocrine manner (Friedman-Einat et al, 1982;Resnitzky et al, 1986;Galabru et al, 1985).…”

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

“…Type I IFNs also may affect hemopoiesis (4)(5)(6): for example, IFN-a/f3 is produced by cultured murine bone marrow (BM) cells in response to colony-stimulating factor 1 (CSF-1) and may suppress or induce differentiation depending on the experimental conditions (6). IFN-a and -13 are constitutively produced by hemopoietic cells (6)(7)(8)(9), but direct evidence of an in vivo role for constitutive IEN in hemopoiesis is lacking. Type I IFNs also may regulate the differentiation of other cell types (2,10).…”